STEAP3 as a biochemical marker of red blood cell storage and toxicity

ABSTRACT

Compositions and methods for determining post-transfusion survival or toxicity of red blood cells and the suitability of red blood cell units for transfusion by measuring the levels of one or more markers in a red blood cell sample are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/308,836, filed on Nov. 3, 2016, which is a U.S. National Phase PatentApplication based on International Patent Application No.PCT/US2015/029552, filed on May 6, 2015, which claims the benefit ofU.S. Provisional Application No. 61/989,440, filed May 6, 2014, all ofwhich are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on May 27, 2015, is namedB212-0027PCT Sequence Listing and is 18,069 bytes in size.

FIELD OF THE INVENTION

The disclosure relates to compositions and methods for determiningpost-transfusion survival and toxicity of red blood cell (RBC) units bymeasuring the levels of one or more markers in a RBC sample.

BACKGROUND OF THE INVENTION

In excess of 15,000,000 units of RBCs are transfused in the USA eachyear into an excess of 5,000,000 patients (approximately 1 out of every65 Americans). Currently, there are only 3 quality control measuresutilized prior to release of a unit of RBCs: 1) testing negative for thescreened pathogens, 2) compatibility with the patient regardingrecipient antibodies to donor antigens, and 3) storage history of 4° C.FDA guidelines for RBC storage require that stored RBCs (up to 42 days)have less than 1% hemolysis and have 75% 24 hour post-transfusionsurvival, on average for a given storage system. However, it has beenappreciated for over forty years that there is tremendous variability inhow individual units of RBCs store from different human donors^(1,2).Even for current blood storage solutions, 24 hour post-transfusionrecoveries range from 35% to 100%² It has been further observed that RBCstorage is reproducible from donation to donation for a givendonor^(3,4), suggesting a potential genetic component^(1,5). Despiteextensive study, there is no measurable entity known to predict how anRBC unit will do when transfused. For this reason, currently, there areno quality control measures (or unit release criteria) regarding qualityof RBC units.

This is a medical problem for a number of reasons. First, RBCs thatsurvive poorly post-transfusion result in a less efficacious productfrom the standpoint of RBC replacement. However, even more important isthe notion that RBCs that are cleared from circulation represent a toxicinsult to the recipient, which may result in morbidity and/or mortality.A second issue is what biochemical markers may predict RBCs that aregoing to be toxic from pathways other than simple RBC clearance.

There are currently no existing techniques to predict post-transfusionsurvival of RBC units or toxicity of said units. Thus, the presentdisclosure satisfies these and other needs. Disclosed herein is a methodfor assessing a RBC unit (prior to transfusion) allowing the predictionof its post-transfusion survival and toxicity. Specifically, biochemicalmarkers that predict if RBCs will survive well post-transfusion or willbe toxic are presented herein.

SUMMARY

Described herein are compositions and methods for determiningpost-transfusion survival and toxicity of a RBC unit by measuring thelevels of one or more markers, including Steap3, in a RBC sample.

In a first aspect, disclosed herein is a method of determiningpost-transfusion survival of red blood cells (RBC) prior to transfusion,the method comprising the steps of: a) providing a sample of RBC; b)measuring the level of activity of Steap3 protein in the RBC sample; c)comparing the level of activity of Steap3 protein in the RBC sample withthe level of Steap3 protein activity in a control RBC sample, wherein ahigher level of activity of Steap3 protein in the RBC sample as comparedto the control sample is indicative of a lower RBC storage quality.

In a second aspect, disclosed herein is a method of determining thesuitability of a red blood cell (RBC) unit for transfusion, the methodcomprising the steps of: a) providing a sample of RBC; b) measuring thelevel of activity of Steap3 protein in the RBC sample; c) comparing thelevel of activity of Steap3 protein in the RBC sample with the level ofSteap3 protein activity in a control sample, wherein a higher level ofactivity of Steap3 protein in the RBC sample is indicative of a lowersuitability for transfusion.

In various embodiments of the first and second aspects, the measurementis performed at the time of collection of the RBC sample.

In various embodiments of the first and second aspects, the measurementis performed during the time of storage of the RBC sample.

In various embodiments of the first and second aspects, the measurementof activity is performed by determining ferric reductase activity.

In various embodiments of the first and second aspects, the level is2-200 fold higher than in the control sample.

In a third aspect, disclosed herein is a method of evaluating thesuitability of a human subject to be blood donor comprising: (a)providing a biological sample from the human subject, wherein the samplecomprises all, or a portion of, an Steap3 gene; and (b) detecting thepresence of a polymorphism in the Steap3 gene or the portion thereof inthe sample, wherein the Steap3 gene comprises at least 90% sequenceidentity to SEQ ID NO: 2; and (c) evaluating the human subject forsuitability as a blood donor based on the presence of a polymorphism inthe Steap3 gene or the portion thereof.

In various embodiments of the third aspect, the polymorphism in theSteap3 gene results in a A>V substitution at position 350 of the proteinsequence.

In various embodiments of the third aspect, the polymorphism in theSteap3 gene results in reduced activity of the Steap3 protein.

In a fourth aspect, disclosed herein is a method of evaluating thesuitability of a human subject to be blood donor comprising: (a)providing a biological sample from the human subject; and (b)determining the level of Steap3 protein activity in the sample; and (c)evaluating the human subject as being an unsuitable blood donor based onthe higher level of Steap3 protein activity in the sample as compared tothe activity in a control sample.

In various embodiments of the fourth aspect, the protein activity isferric reductase activity.

In a fifth aspect, disclosed herein is a method for determining RBCstorage quality, the method comprising the steps of: obtaining a datasetassociated with a sample of stored blood, wherein the dataset comprisesa Steap3 marker; analyzing the dataset to determine data for the Steap3marker, wherein the data is positively correlated or negativelycorrelated with RBC storage quality of the sample of stored blood.

In various embodiments of the fifth aspect, the data is protein level,mRNA level, protein activity level, or sequence of the Steap3 protein ornucleic acid.

In a sixth aspect, disclosed herein is a method for determining RBCstorage quality, the method comprising the steps of: providing a sampleof stored blood, wherein the sample comprises a Steap3 marker;contacting the sample with a reagent; generating a complex between thereagent and the Steap3 marker; detecting the complex to obtain a datasetassociated with the sample, wherein the dataset comprises expression oractivity level or sequence data for the Steap3 marker; and analyzing theexpression or activity level data for the a Steap3 marker, wherein theexpression or activity level or sequence of the a Steap3 marker ispositively correlated or negatively correlated with RBC storage quality.

In a seventh aspect, disclosed herein is a computer-implemented methodfor determining RBC storage quality, the method comprising the steps of:storing, in a storage memory, a dataset associated with a stored bloodsample, wherein the dataset comprises data for a Steap3 marker; andanalyzing, by a computer processor, the dataset to determine theexpression or activity levels or sequence of the Steap3 marker, whereinthe expression or activity levels or sequence are positively correlatedor negatively correlated with RBC storage quality.

In an eighth aspect, disclosed herein is a system for determining RBCstorage quality, the system comprising: a storage memory for storing adataset associated with a stored blood sample, wherein the datasetcomprises data for a Steap3 marker; and a processor communicativelycoupled to the storage memory for analyzing the dataset to determine theactivity or expression levels or sequence of the Steap3 marker, whereinthe activity or expression levels or sequence are positively correlatedor negatively correlated with RBC storage quality.

In a ninth aspect, disclosed herein is a computer-readable storagemedium storing computer-executable program code, the program codecomprising: program code for storing a dataset associated with a storedblood sample, wherein the dataset comprises data for a Steap3 marker;and program code for analyzing the dataset to determine the activity orexpression levels or sequence of the Steap3 marker, wherein the activityor expression levels or sequence of the markers are positivelycorrelated or negatively correlated with RBC storage quality.

In a tenth aspect, disclosed herein is a method for predicting anegative transfusion outcome, the method comprising the steps of:obtaining a dataset associated with a sample of stored blood, whereinthe dataset comprises data for a Steap3 marker; analyzing the dataset todetermine data for the at least one marker, wherein the data ispositively correlated or negatively correlated with a negativetransfusion outcome if the blood sample is transfused into a patient.

In an embodiment of the tenth aspect, the data is protein level, mRNAlevel, protein activity level, or sequence of the Steap3 protein ornucleic acid.

In an eleventh aspect, disclosed herein is a method for predicting anegative transfusion outcome, the method comprising the steps of:providing a sample of stored blood, wherein the sample comprises aSteap3 marker; contacting the sample with a reagent; generating acomplex between the reagent and the Steap3 marker; detecting the complexto obtain a dataset associated with the sample, wherein the datasetcomprises expression or activity level or sequence data for the Steap3marker; and analyzing the expression or activity level data for themarkers, wherein the expression or activity level or sequence of the atleast one marker is positively correlated or negatively correlated witha negative transfusion outcome if the blood sample is transfused into apatient.

In a twelfth aspect, disclosed herein is a computer-implemented methodfor predicting a negative transfusion outcome, the method comprising thesteps of: storing, in a storage memory, a dataset associated with astored blood sample, wherein the dataset comprises data for a Steap3marker; and analyzing, by a computer processor, the dataset to determinethe expression or activity levels or sequence of the Steap3 marker,wherein the expression or activity levels or sequence are positivelycorrelated or negatively correlated with a negative transfusion outcomeif the blood sample is transfused into a patient.

In a thirteenth aspect, disclosed herein is a system for predicting anegative transfusion outcome, the system comprising: a storage memoryfor storing a dataset associated with a stored blood sample, wherein thedataset comprises data for a Steap3 marker; and a processorcommunicatively coupled to the storage memory for analyzing the datasetto determine the activity or expression levels or sequence of the Steap3marker, wherein the activity or expression levels or sequence arepositively correlated or negatively correlated with a negativetransfusion outcome if the blood sample is transfused into a patient.

In a fourteenth aspect, disclosed herein is a computer-readable storagemedium storing computer-executable program code, the program codecomprising: program code for storing a dataset associated with a storedblood sample, wherein the dataset comprises data for a Steap3 marker;and program code for analyzing the dataset to determine the activity orexpression levels of the Steap3 marker, wherein the activity orexpression levels or sequence of the markers are positively correlatedor negatively correlated with a negative transfusion outcome if theblood sample is transfused into a patient.

In embodiments of the method or storage medium or system of the aspectsabove, the dataset is obtained at the time of collection of the RBCsample.

The method or storage medium or system of any one of the aspects above,wherein the dataset is obtained during the time of storage of the RBCsample.

In embodiments of the method or storage medium or system of the aspectsabove, the dataset is obtained by determining ferric reductase activity.

In a fifteenth aspect, disclosed herein is a method for determiningpost-transfusion survival of red blood cells (RBC) prior to transfusioncomprising: a) generating data on the level of Steap3 in a sample fromthe subject; b) generating data on the level of at least one additionalbiomarker or indicator of blood storage c) generating a score bymathematically combining the data in (a) and (b), wherein the score isindicative of post-transfusion survival of red blood cells (RBC) in thesample.

In an embodiment of the fifteenth aspect, the score is used to determinewhether the RBC sample will be administered to the subject.

In an embodiment of the fifteenth aspect, the score is generated by acomputer processor.

In various embodiments of the above, the method further comprises thestep of administering or not administering the RBC sample that has beentested.

In a sixteenth aspect, disclosed herein is a kit for use in predicting anegative transfusion outcome or red blood cell (RBC) storage quality,the kit comprising: a set of reagents comprising a plurality of reagentsfor determining from a stored blood sample data for Steap3 marker; andinstructions for using the plurality of reagents to determine data fromthe stored blood sample.

In various embodiments of the sixteenth aspect, data is protein level,mRNA level, protein activity level, or sequence of the Steap3 protein ornucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows the correlation between Steap3 activity and RBCstorage. (FIG. 1A) Each strain had slightly different RBC storagecharacteristics, as measured by post-transfusion 24-hour recoveries ofstored RBCs. (FIG. 1B) 6 particular stains, ranging in storage, werechosen for further analysis by ferrozine assay (measuring Steap3activity). (FIG. 10) A Pearson's correlation coefficient of −0.7185 wasobserved between the ferrozine activity and the 24-hr RBC recoveries.

DETAILED DESCRIPTION

The present invention generally relates to compositions and methods fordetermining post-transfusion survival and toxicity of RBCs by measuringthe levels of one or more markers in a RBC sample.

The invention described in this disclosure represents a method forassessing an RBC unit (prior to transfusion) allowing the prediction ofits post-transfusion survival and toxicity. Among the specific aims are:(1) Biochemical markers that predict if RBCs will survive wellpost-transfusion; and (2) Biochemical markers that predict if RBCs aretoxic post-transfusion.

Red blood cell (RBC) transfusion is a life-saving therapy, andrefrigerated storage is crucial for maintaining an adequate supply ofdonor units. However, recent studies have focused on potential adverseclinical sequelae resulting from transfusing humans with RBC unitsstored for longer periods of time. Indeed, multiple observationalstudies in human patients provide data demonstrating inferior clinicaloutcomes when older, stored RBC units are transfused¹. Nonetheless, thisissue remains controversial because other, similarly designed humanstudies, show no difference in clinical outcome when comparing patientsreceiving transfusions of older or fresher RBC units^(1,2). To begin toaddress this controversy, several prospective human trials are currentlyongoing, and one was recently completed³⁻⁵. However, it is notcontroversial that stored RBCs accumulate multiple factors that may betoxic when infused (e.g. microparticles, free iron, free hemoglobin,prostaglandins, and leukotrienes)⁶⁻¹⁴.

<See Ref List Below>

One complication in studying RBC transfusion is that there isconsiderable donor-to-donor variation in the effect of refrigeratedstorage on RBC function and quality. In addition, there is a generalabsence of robust analytic tests that consistently and accuratelypredict the quality of a given RBC unit prior to transfusion¹⁵. Due tothe genetic and environmental complexity of outbred human donorpopulations, and the difficulty in limiting the number of independentvariables in studying human RBC transfusion, we developed a robustanimal model to begin to address these issues. Using inbred mousestrains in defined environmental and dietary settings limits theexperimental variability of the system, and allows for deliberatemanipulation of independent variables.

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. As used in this specification andthe appended claims, the singular forms “a”, “an” and “the” includeplural references unless the content clearly dictates otherwise.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

As used herein, “RBC storage quality” is defined as the extent ofpost-transfusion recovery of the stored RBCs; higher recovery is definedas higher quality. Examples of post-transfusion recovery include greaterthan zero and almost 100% recovery, i.e., recovery of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, and all percentages in between. Inone embodiment, an acceptable RBC storage quality is an average of 75%post-transfusion recovery at 24 hours, as under FDA guidelines.

As used herein, “toxicity” of a RBC unit is defined as any adversereaction associated with transfusion of a RBC unit, including, but notlimited to, hemolytic transfusion reactions, exposure to freehemoglobin, iron overload, induction of recipient cytokines,introduction of procoagulant activity, and inhibition of recipientvascular relaxation, among others.

As used herein, a RBC unit is less suitable for transfusion if it haslower RBC quality (i.e., lower post-transfusion survival) or elevatedtoxicity as compared to other RBC units, e.g., as compared to a control.

An “analyte” or “target” refers to a compound to be detected. Suchcompounds can include small molecules, peptides, proteins, nucleicacids, as well as other chemical entities. In the context of the presentinvention, an analyte or target will generally correspond to thebiochemical compounds disclosed herein, or a reaction product thereof.

To “analyze” includes determining a value or set of values associatedwith a sample by measurement of analyte levels in the sample. “Analyze”may further comprise comparing the levels against constituent levels ina sample or set of samples from the same subject or other subject(s).For example, the blood storage markers, such as Steap3, of the presentteachings can be analyzed by any of various conventional methods knownin the art.

The term “biomarker” refers to a molecule (typically small molecule,protein, nucleic acid, carbohydrate, or lipid) that is expressed and/orreleased from a cell, which is useful for identification or prediction.Such biomarkers are molecules that can be differentially expressed,e.g., overexpressed or underexpressed, or differentially released inresponse to varying conditions (e.g., storage). In the context of thepresent invention, this frequently refers to Steap3 as disclosed herein,which shows altered levels of activity in stored versus non-stored RBCs,for instance, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold or more in storedRBCs versus non-stored RBCs.

A “sample” refers to any source which is suspected of containing ananalyte or target molecule. Examples of samples which may be testedusing the present invention include, but are not limited to, blood,serum, plasma, urine, saliva, cerebrospinal fluid, lymph fluids, tissueand tissue and cell extracts, cell culture supernantants, among others.A sample can be suspended or dissolved in liquid materials such asbuffers, extractants, solvents, and the like. In the context of thepresent application, a sample is generally a stored RBC sample ofvarying length of storage.

The term “subject” encompasses a cell, tissue, or organism, human ornon-human, whether in vivo, ex vivo, or in vitro, male or female.

The term “generating data” encompasses obtaining a set of datadetermined from at least one sample. Generating data encompassesobtaining a sample, and processing the sample to experimentallydetermine the data. The phrase also encompasses receiving a set of data,e.g., from a third party that has processed the sample to experimentallydetermine the data. Additionally, the phrase encompasses mining datafrom at least one database or at least one publication or a combinationof databases and publications. Data can be obtained by one of skill inthe art via a variety of known ways including stored on a storagememory. Obtaining data encompasses data which has been generated from asample, or data which has been obtained from sources such as patientmedical history and records, physical examinations, treatment history,and the like.

The term “clinical factor” refers to a measure of a condition of asubject, e.g., disease activity or severity. “Clinical factor”encompasses all indicators of a subject's health status, includingnon-sample markers, and/or other characteristics of a subject, such as,without limitation, age and gender. A clinical factor can be a score, avalue, or a set of values that can be obtained from evaluation of asample (or population of samples) from a subject or a subject under adetermined condition.

The term “algorithm” encompasses any formula, model, mathematicalequation, algorithmic, analytical or programmed process, or statisticaltechnique or classification analysis that takes one or more inputs orparameters, whether continuous or categorical, and calculates an outputvalue, index, index value or score. Examples of algorithms include butare not limited to ratios, sums, regression operators such as exponentsor coefficients, biomarker value transformations and normalizations(including, without limitation, normalization schemes that are based onclinical parameters such as age, gender, ethnicity, etc.), rules andguidelines, statistical classification models, and neural networkstrained on populations. Also of use in the context of blood storagefactors are linear and non-linear equations and statisticalclassification analyses to determine the relationship between (a) levelsof blood storage markers, such as Steap3, detected in a subject sampleand (b) the level of blood storage or blood quality from the respectivesubject.

“Antibody” refers to any immunoglobulin or intact molecule as well as tofragments thereof that bind to a specific epitope that may be used inthe practice of the present invention. Such antibodies include, but arenot limited to polyclonal, monoclonal, chimeric, humanized, singlechain, Fab, Fab′, F(ab)′ fragments and/or F(v) portions of the wholeantibody and variants thereof. All isotypes are encompassed by this termand may be used in the practice of this invention, including IgA, IgD,IgE, IgG, and IgM.

An “antibody fragment” refers specifically to an incomplete or isolatedportion of the full sequence of the antibody which retains the antigenbinding function of the parent antibody and may also be used in thepresent invention. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

An intact “antibody” for use in the invention comprises at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH₁, CH₂and CH₃. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region is comprised of one domain, C_(L). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxyl-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies can mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term antibodyincludes antigen-binding portions of an intact antibody that retaincapacity to bind. Examples of binding include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature, 341:544-546 (1989)),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR).

“Single chain antibodies” or “single chain Fv (scFv)” may also be usedin the present invention. This term refers to an antibody fusionmolecule of the two domains of the Fv fragment, V_(L) and V_(H).Although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see, e.g., Bird et al.,Science, 242:423-426 (1988); and Huston et al., Proc Natl Acad Sci USA,85:5879-5883 (1988)). Such single chain antibodies are included byreference to the term “antibody” fragments can be prepared byrecombinant techniques or enzymatic or chemical cleavage of intactantibodies.

A “monoclonal antibody” may be used in the present invention. Monoclonalantibodies are a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope.

In one embodiment, the antibody or fragment is conjugated to an“effector” moiety. The effector moiety can be any number of molecules,including labeling moieties such as radioactive labels or fluorescentlabels.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

Samples of RBCs stored for various amounts of time are compared to“control” samples which can be freshly drawn RBCs or RBCs which havebeen minimally stored. Control samples are assigned a relative analyteamount or activity to which sample values are compared. Relevant levelsof analyte elevation occur when the sample amount or activity valuerelative to the control is 110%, more preferably 150%, more preferably200-500% (i.e., two to five fold higher relative to the control), morepreferably 1000-3000% higher. Alternatively, relevant levels of analytedecrease occur when the sample amount or activity value relative to thecontrol is 90%, more preferably 50%, more preferably 20-50% (i.e., twoto five fold lower relative to the control), more preferably 20-100%lower.

Assays for many of the biochemical compounds disclosed herein are knownor commercially available.

For example, antibody reagents can be used in assays to detect thelevels of analytes in RBC samples using any of a number of immunoassaysknown to those skilled in the art.

Immunoassay techniques and protocols are generally described in Priceand Newman, “Principles and Practice of Immunoassay,” 2nd Edition,Grove's Dictionaries, 1997; and Gosling, “Immunoassays: A PracticalApproach,” Oxford University Press, 2000. A variety of immunoassaytechniques, including competitive and non-competitive immunoassays, canbe used. See, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65(1996). The term immunoassay encompasses techniques including, withoutlimitation, enzyme immunoassays (EIA) such as enzyme multipliedimmunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA),IgM antibody capture ELISA (MAC ELISA), and microparticle enzymeimmunoassay (META); immunohistochemical (IHC) assays; capillaryelectrophoresis immunoassays (CEIA); radioimmunoassays (RIA);immunoradiometric assays (IRMA); fluorescence polarization immunoassays(FPIA); and chemiluminescence assays (CL). If desired, such immunoassayscan be automated. Immunoassays can also be used in conjunction withlaser induced fluorescence. See, e.g., Schmalzing et al.,Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci.,699:463-80 (1997). Liposome immunoassays, such as flow-injectionliposome immunoassays and liposome immunosensors, are also suitable foruse in the present invention. See, e.g., Rongen et al., J. Immunol.Methods, 204:105-133 (1997). In addition, nephelometry assays, in whichthe formation of protein/antibody complexes results in increased lightscatter that is converted to a peak rate signal as a function of themarker concentration, are suitable for use in the methods of the presentinvention. Nephelometry assays are commercially available from BeckmanCoulter (Brea, Calif.; Kit #449430) and can be performed using a BehringNephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem.,27:261-276 (1989)).

Specific immunological binding of the antibody to proteins can bedetected directly or indirectly. Direct labels include fluorescent orluminescent tags, metals, dyes, radionuclides, and the like, attached tothe antibody. A chemiluminescence assay using a chemiluminescentantibody specific for the protein is suitable for sensitive,non-radioactive detection of protein levels. An antibody labeled withfluorochrome is also suitable. Examples of fluorochromes include,without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin,B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine.Indirect labels include various enzymes well known in the art, such ashorseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, urease, and the like. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable at 450 nm. Analkaline phosphatase detection system can be used with the chromogenicsubstrate p-nitrophenyl phosphate, for example, which yields a solubleproduct readily detectable at 405 nm. Similarly, a β-galactosidasedetection system can be used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a solubleproduct detectable at 410 nm. An urease detection system can be usedwith a substrate such as urea-bromocresol purple (Sigma Immunochemicals;St. Louis, Mo.).

A signal from the direct or indirect label can be analyzed, for example,using a spectrophotometer to detect color from a chromogenic substrate;a radiation counter to detect radiation such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. For detection ofenzyme-linked antibodies, a quantitative analysis can be made using aspectrophotometer such as an EMAX Microplate Reader (Molecular Devices;Menlo Park, Calif.) in accordance with the manufacturer's instructions.If desired, the assays of the present invention can be automated orperformed robotically, and the signal from multiple samples can bedetected simultaneously.

The antibodies can be immobilized onto a variety of solid supports, suchas magnetic or chromatographic matrix particles, the surface of an assayplate (e.g., microtiter wells), pieces of a solid substrate material ormembrane (e.g., plastic, nylon, paper), and the like. An assay strip canbe prepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

In some embodiments, the measurement of the markers of the presentinvention is performed using various mass spectrometry methods. As usedherein, the term “mass spectrometry” or “MS” refers to an analyticaltechnique to identify compounds by their mass. MS refers to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z”. MS technology generally includes (1) ionizing thecompounds to form charged compounds; and (2) detecting the molecularweight of the charged compounds and calculating a mass-to-charge ratio.The compounds may be ionized and detected by any suitable means. A “massspectrometer” generally includes an ionizer and an ion detector. Ingeneral, one or more molecules of interest are ionized, and the ions aresubsequently introduced into a mass spectrographic instrument where, dueto a combination of magnetic and electric fields, the ions follow a pathin space that is dependent upon mass (“m”) and charge (“z”). See, e.g.,U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;”U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics BasedOn Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21; 1164-67.

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

Steap3

The Steap3 gene encodes a metalloreductase with the activity ofcoverting iron from an insoluble ferric (Fe³⁺) to a soluble ferrous(Fe²⁺) form.

Reference sequences for the mouse gene and protein have the NCBIaccession numbers NM_001085409 and NP_001078878. The human gene andprotein have the NCBI accession number NM_001008410. Representativesequences of Steap3 genes and proteins are shown below.

TABLE 1 Accession SEQ Number And ID NO DescriptionAmino acid/nucleotide sequence 1 NM_001008MPEEMDKPLISLHLVDSDSSLAKVPDEAPKVGILGSGDFARSLATRLVGSGFKVVVGSRNPKRT 410ARLFPSAAQVTFQEEAVSSPEVIFVAVFREHYSSLCSLSDQLAGKILVDVSNPTEQEHLQHRES HumanNAEYLASLFPTCTVVKAFNVISAWTLQAGPRDGNRQVPICGDQPEAKRAVSEMALAMGFMPVDM proteinGSLASAWEVEAMPLRLLPAWKVPTLLALGLFVCFYAYNFVRDVLQPYVQESQNKFFKLPVSVVNTTLPCVAYVLLSLVYLPGVLAAALQLRRGTKYQRFPDWLDHWLQHRKQIGLLSFFCAALHALYSFCLPLRRAHRYDLVNLAVKQVLANKSHLWVEEEVWRMEIYLSLGVLALGTLSLLAVTSLPSIANSLNWREFSFVQSSLGFVALVLSTLHTLTYGWTRAFEESRYKFYLPPTFTLTLLVPCVVILAKALFLLPCISRRLARIRRGWERESTIKFTLPTDHALAEKTSHV 2 NM_001008gaggaggagc ctcgggccga gccaccgcct tcgccgcgga ccttcagctg 410ccgcggtcgctccgagcggc gggccgcaga gccaccaaaa tgccagaaga gatggacaag Humanccactgatcagcctccacct ggtggacagc gatagtagcc ttgccaaggt ccccgatgagnucleotidegcccccaaagtgggcatcct gggtagcggg gactttgccc gctccctggc cacacgcctggtgggctctggcttcaaagt ggtggtgggg agccgcaacc ccaaacgcac agccaggctgtttccctcagcggcccaagt gactttccaa gaggaggcag tgagctcccc ggaggtcatctttgtggctgtgttccggga gcactactct tcactgtgca gtctcagtga ccagctggcgggcaagatcctggtggatgt gagcaaccct acagagcaag agcaccttca gcatcgtgagtccaatgctgagtacctggc ctccctcttc cccacttgca cagtggtcaa ggccttcaatgtcatctctgcctggaccct gcaggctggc ccaagggatg gtaacaggca ggtgcccatctgcggtgaccagccagaagc caagcgtgct gtctcggaga tggcgctcgc catgggcttcatgcccgtggacatgggatc cctggcgtca gcctgggagg tggaggccat gcccctgcgcctcctcccggcctggaaggt gcccaccctg ctggccctgg ggctcttcgt ctgcttctatgcctacaacttcgtccggga cgttctgcag ccctatgtgc aggaaagcca gaacaagttcttcaagctgcccgtgtccgt ggtcaacacc acactgccgt gcgtggccta cgtgctgctgtcactcgtgtacttgcccgg cgtgctggcg gctgccctgc agctgcggcg cggcaccaagtaccagcgcttccccgactg gctggaccac tggctacagc accgcaagca gatcgggctgctcagcttcttctgcgccgc cctgcacgcc ctctacagct tctgcttgcc gctgcgccgcgcccaccgctacgacctggt caacctggca gtcaagcagg tcttggccaa caagagccacctctgggtggaggaggaggt ctggcggatg gagatctacc tctccctggg agtgctggccctcggcacgttgtccctgct ggccgtgacc tcactgccgt ccattgcaaa ctcgctcaactggagggagttcagcttcgt tcagtcctca ctgggctttg tggccctcgt gctgagcacactgcacacgctcacctacgg ctggacccgc gccttcgagg agagccgcta caagttctacctgcctcccaccttcacgct cacgctgctg gtgccctgcg tcgtcatcct ggccaaagccctgtttctcctgccctgcat cagccgcaga ctcgccagga tccggagagg ctgggagagggagagcaccatcaagttcac gctgcccaca gaccacgccc tggccgagaa gacgagccacgtatgaggtgcctgccctgg gctctggacc ccgggcacac gagggacggt gccctgagcccgttaggttttcttttcttg gtggtgcaaa gtggtataac tgtgtgcaaa taggaggtttgaggtccaaattcctgggac tcaaatgtat gcagtactat tcagaatgat atacacacatatgtgtatatgtatttacat atattccaca tatataacag gatttgcaat tatacatagctagctaaaaagttgggtctc tgagatttca acttgtagat ttaaaaacaa gtgccgtacgttaagagaaggcagatcat gctattgtga catttgcaga gatatacaca cactttttgtacagaagaggcttgtgctgt ggtgggttcg atttatccct gcccacccca cccccacaacttcccttttgctacttcccc aaggctcttg cagagctagg gctctgaagg ggagggaaggcaacggctctgcccagagcc atccctggag catgtgagca gcggctggtc tcttccctccacctggggcagcagcaggag gcctggggag gaggaaaatc aggcagtcgg cctggagtctgtgcctggtcctttgcccgg tggtgggagg atggagggat tgggctgaag ctgctccacctcatccttgctgagtggggg agacattttc cctgaaagtc agaagtcacc atagagcctgcaaatggatcctcctgtgag agtgacgtca cctcctttcc agagccatta gtgagcctggcttgggaacaagtgtaattt ccttccctcc tttaacctgg cgatgagcgt cctttaaaccactgtgccttctcacccttt ccatcttcag tttgaatgac tcccaggaag gcctagagcagaccctttagaaatcagccc aagggggaga gcaagagaaa acactctagg gagtaaagctccccgggcgtcagagttgag ccctgcctgg gctgaaggac tgtcttcacg aagtcagtcctgaggaaaaatattggggac tccaaatgtc ctctggcaga ggacccagaa aaccacactggctccaacttcctcctcatg gggcattaca cttcaaaaca gtggggagca acttttccaccaaagctacaaacctaaaat gctgctgccc caaagcacaa gagggaagag caccgccggggccacaggacgtctgtcctc cagtcacagg ccatccttgc tgctccctac tgactctagcttacttcccctgtgaagaaa caggtgttct cggctgagcc cccaaccctc tgcagaaccaggttgatctgccacagaaaa agcatctttg aagacaaaga gggtgaggtc ttcatgagtctcctgggcccaaagccatct tctgatgga ggaagagagt agggccagtg aaggctgcccagagagaatgtcacagatga ggctgcccct gccccccccc cgccagggag gtttcatgagctcatgtctatgcagcacat aagggttctt cagtgaaaag caggagaaga gcccactgcaaggatagctcattaggcaca tgaccgatgc agggaaggcc atgccgggga agctcttcctgcaggtattttccatctgct gtgccaaggc tgagcggcag aaacttgtct cataaattggcactgatggagcatcagctg tggcccacag agagccttgc tgagaagggg gcaggtaaagcagagattttagcattgcct tggcataaca agggcccatc gattccctac taatgagaggcagggagagcatgggcaatg gagacccacc aatgatcccc aaccccggtg ggtactggctgcctgccctgggccagggaa tggctcctta taccaaagat gctggcacat agcagaacccagtgcacgtcctccccttcc cacccacctc tggctgaagg tgctcaagag ggaagcaattataaggtgggtggcaggagg gaacaggtgc cacctgctgg acaatcacac gaaaggcaggcgggctgtgtactgggccct gactgtgcgt ccactgctgt cttccctacc tcaccaggctactggcagcagcatcccgag agcacatcat ctccacagcc tggtaaattc catgtgcctctgggtacaaaagtgcctcaa cgacatgctc tggaaatccc aaatgccaca gtctgaggttgatatctaaaatctatgcct tcaaaagagt ctctgttttt tttttttaac ctggtagacagtataaaagcagtgcaaata aacacctaac cttctgcaaa 3 NP_001078MSGEMDKPLI SRRLVDSDGS LAEVPKEAPK VGILGSGDFA RSLATRLVGS 878GFSVVVGSRNPKRTAGLFPS LAQVTFQEEA VSSPEVIFVA VFREHYSSLC SLADQLAGKI MouseLVDVSNPTEKEHLQHRQSNA EYLASLFPAC TVVKAFNVIS AWALQAGPRD GNRQVLICSD proteinQPEAKRTISEMARAMGFTPL DMGSLASARE VEAIPLRLLP SWKVPTLLAL GLFVCFYTYNFIRDVLQPYIRKDENKFYKM PLSVVNTTLP CVAYVLLSLV YLPGVLAAAL QLRRGTKYQRFPDWLDHWLQHRKQIGLLSF FFAMLHALYS FCLPLRRSHR YDLVNLAVKQ VLANKSRLWAEEEVWRMEIYLSLGVLALGM LSLLAVTSLP SIANSLNWKE FSFVQSTLGF VALILSTMHTLTYGWTRAFEENHYKFYLPP TFTLTLLLPC VIILAKGLFL LPCLNRRLTK IRRGWEKDGAVKFMLPGDHTQGEKTSHV 4 NM_001085tcacactcta tctcagacct tgagctaaag gactctccca aggcgagggg 409gccactgcttactggggccc tccacctggg aaggagaggt taaaggtgac ccagggttaa Mouseggagaacctggtatgccaca tctcaactta tgatgtgagc tccagccacc cagcttcaganucleotideagaaatggctgcagaggccc acaggcagca gggctcttgc cccaccatcc cctcagagggctgtggaaagtcaccagaga agaaaggcag tgcggccgac tctagacctg gtgagctggagtctgtggggcagggaggaa gcagggcata gaagacacag gaagcaactc tgccctgattccagactccatctgcatgga ctgattccag gtgctagggt tcctttctca gaaaccccagaagtccacgaaggcactgct atgtcggggg agatggacaa gccgctgatc agccgccgcctagtggacagtgatggcagt ctggctgagg tccccaagga ggcccccaaa gtgggcatcctgggcagtggggattttgcc cgttccctgg ccacacgcct ggtgggctct ggcttcagtgtggtggtggggagccgtaac cccaaacgca cggctggcct cttcccctcc ttagctcaagtgactttccaggaggaagcc gtgagctctc cagaggtcat ctttgtggcc gtgttccgggagcactattcctcactgtgc agtctcgctg accagttggc tggcaagatc ctcgtggatgtaagcaaccccacggagaag gagcatcttc agcaccgcca gtctaacgct gagtacctggcctcactctttcctgcgtgc actgtggtga aggccttcaa cgtcatctct gcatgggccctacaggctggcccaagggat gggaacaggc aggtgctcat ctgcagtgat cagccagaagccaagcgcaccatctcagag atggcacgcg ccatgggttt cacacccctg gacatgggatccctggcctcagcgagggag gtagaagcca tacccctgcg cctccttcca tcctggaaggtgcccaccctcctggcactg gggctctttg tgtgcttcta cacctacaac ttcatccgag acgttctacagccatacatt cggaaagatg agaacaagtt ctacaagatg cccttgtctgtggtcaacaccacactaccc tgtgtggctt atgtgctgct gtccctagtg tacctgcccggtgtgctggcagctgcgctt cagctgcgga gggggaccaa gtaccagcgc ttcccagactggctggaccactggctgcag catcgcaagc agatcgggct gctcagcttc ttcttcgcgatgctgcacgctctctacagc ttctgcctgc cgctgcgccg ctcccaccgc tacgacctggtcaatctggctgtgaagcag gtcctggcca acaagagccg cctctgggct gaggaagaagtctggaggatggagatatac ctgtccctgg gtgtgctggc cctgggcatg ttgtcgctgctggctgtcacctcgctcccg tccattgcta attccctcaa ctggaaggag ttcagcttcgtgcagtccacactgggcttc gtggccctga tactcagcac aatgcacaca ctcacctacggctggacccgtgcctttgag gaaaaccact acaagttcta cctgccgccc acattcacactcacgctgctcctgccctgt gtgatcatcc tggccaaggg cctcttcct ctgccctgcctcaaccgcagactcaccaag atacgcaggg gctgggagaa agatggggct gtcaagttcatgctgcccggcgaccacaca cagggggaga aaacaagcca cgtgtgaggc cctggaagtggagatggcttgtgggggccc tgagctgggt tcgggtctct tttctggatg ctgcacagcgaggtgatgatatatgcgtgg gtggctgaga tcctaattcc tgggatgcag gtgtaaactgacatactcagaatgacaccc catacatgtg atatgtactt acatatattt cacatataataagatttgctattattctta cttagctaaa aaaaaaaagt gggtccctat atttcagcgtaagcatttcaaagcaaatgc cacacattga acagcagatc ccacccttgt ggtatctacagaggcagacagacactctgg tataggagaa actgtctttc gttggattct ctcctttaatctctatgctccttattagct gaatcctaaa gttggtgcaa agctggggca agaaatgcctctggtgccgcctacccccat cccagggcta agaaagaagc ctcgagtgaa cagggaaccaggtctggactctgctgcttc cctgggcgtg cgtggggagg ctcagcaaga ccccctgggatctatgcaggagctttttca ggtccgtcct ttcttcaggg aagggtctga agctgccccatctgatcctagctgagctga gaagattctt ccccaccccc tgaaagtcca gagtcaccaccggagcctgcaaattgatcc ttctgcgaag gtgtgaagtc accgcctctc cagagccattaatgaacctggtcttcggga ggaggataat tgtttcctct ccattaagtt gctggtgaccccccctttaaatcactgtgc cttctcgcct tttccatcat taatttggac atctccgtggagtggacacttgtctgggca gtccggggtg ggggggagca ttagagattg cagagaataaccatcgaatcctcttcttgg ggcaaccctc cccttggatg tgccccaggc ctgccttcattaaattggtccctgaggaga ataataggga cccttttcat ttaccctgtc gcctgtaggcagaaaacctaccttctgagc acccagaaaa cacagtggcc ccatgctctt cttcagggggttccacagcccccttccccg tgtttttgcc tccctccctc cttcctcccc tccctccctccctcactgttacgttcaacc acaaaagtct tcaaatattg tttttttgaa ttcttaaagagacctcattttattacaaaa aaaaaaaaaa aa

The sequence of Steap3 indicates that it is a six-transmembrane protein.

The quantity of Steap3 of the present teachings can be indicated as avalue. The value can be one or more numerical values resulting from theevaluation of a sample, and can be derived, e.g., by measuring level(s)of Steap3 in a sample by an assay performed in a laboratory, or fromdata obtained from a provider such as a laboratory, or from data storedon a server. Steap3 levels can be measured using any of severaltechniques known in the art, such as those described herein.

The measurement of levels of Steap3 can be determined at the protein ornucleic acid level using any method known in the art. “Protein”detection comprises detection of full-length proteins, mature proteins,pre-proteins, polypeptides, isoforms, mutations, variants,post-translationally modified proteins and variants thereof, and can bedetected in any suitable manner. Levels of Steap3 can be determined atthe protein level directly or by measuring the enzymatic activitiesSteap3. Such methods are well-known in the art and include, e.g.,immunoassays based on antibodies to proteins encoded by the genes,aptamers or molecular imprints.

Steap3 can also be determined at the nucleic acid level. For example,nucleic acid sequences that correspond Steap3 can be used to constructprimers and probes for detecting and/or measuring Steap3 nucleic acids.These probes can be used in, e.g., Northern or Southern blothybridization analyses, ribonuclease protection assays, and/or methodsthat quantitatively amplify specific nucleic acid sequences. As anotherexample, Steap3 sequences can be used to construct primers forspecifically amplifying Steap3 sequences in, e.g., amplification-baseddetection and quantitation methods such as reverse-transcription basedpolymerase chain reaction (RT-PCR) and PCR. When alterations in geneexpression are associated with gene amplification, nucleotide deletions,polymorphisms, post-translational modifications and/or mutations,sequence comparisons in test and reference populations can be made bycomparing relative amounts of the examined DNA sequences in the test andreference populations.

As an example, Northern hybridization analysis using probes whichspecifically recognize one or more of these sequences can be used todetermine gene expression. Alternatively, expression can be measuredusing RT-PCR; e.g., polynucleotide primers specific for thedifferentially expressed Steap3 mRNA sequences reverse-transcribe themRNA into DNA, which is then amplified in PCR and can be visualized andquantified. Steap3 RNA can also be quantified using, for example, othertarget amplification methods, such as TMA, SDA, and NASBA, or signalamplification methods (e.g., bDNA), and the like. Ribonucleaseprotection assays can also be used, using probes that specificallyrecognize Steap3 mRNA sequences, to determine gene expression.

In some embodiments, Steap3 is detected by contacting a subject samplewith reagents, generating complexes of reagent and analyte, anddetecting the complexes. Examples of “reagents” include but are notlimited to nucleic acid primers, peptides, small molecules, substratesfor the enzyme, and antibodies.

Various methods to detect Steap3 levels are known in the art. Forexample, various PCR based assays such as TaqMan assays are known in theart and are commercially available.

The sequence of Steap3 can be determined using any of the methods fornucleic acid sequence determination known in the art, such asconventional Sanger sequencing or NextGen sequencing methods.

At the protein level, various antibodies directed against the Steap3protein are known in the art and are commercially available. Steap3ELISA kits are also commercially available.

Assays to measure Steap3 activity are also known. For example, an assaythat measures the production of ferrous iron using 200 μM ferrozine asan indicator and monitoring the increase in absorbance at λ=562 nm maybe used. See, e.g., Ohgami et al., “The Steap proteins aremetalloreductases”, Blood, 108: 1388-1394 (2006).

In some embodiments, data on the level or activity or sequence of Steap3may be combined with other indicators or biomarkers of blood storage,such as those disclosed in U.S. Patent Application No. 2014/0178904, toobtain a score which is indicative of blood storage quality.

The information from the assays above can be quantitative and sent to acomputer system of the invention. The information can also bequalitative, such as observing patterns or fluorescence, which can betranslated into a quantitative measure by a user or automatically by areader or computer system. In an embodiment, the subject can alsoprovide information other than assay information to a computer system,such as race, height, weight, age, gender, eye color, hair color, familymedical history and any other information that may be useful to a user,such as a clinical factor described above.

Other embodiments of the present teachings comprise Steap3 detectionreagents packaged together in the form of a kit for conducting any ofthe assays of the present teachings. In certain embodiments, the kitscomprise reagents for protein detection of Steap3 proteins, such asantibodies. For example, the kit may comprise antibodies or fragmentsthereof, specific for Steap3 (primary antibodies), along with one ormore secondary antibodies that may incorporate a detectable label; suchantibodies may be used in an assay such as an ELISA. Alternately, theantibodies or fragments thereof may be fixed to a solid surface, e.g. anantibody array. In certain embodiments, the kits compriseoligonucleotides that specifically identify one or more Steap3 nucleicacids based on homology and/or complementarity with Steap3 nucleicacids. The oligonucleotide sequences may correspond to fragments ofSteap3 nucleic acids. For example, the oligonucleotides can be more than200, 200, 150, 100, 50, 25, 10, or fewer than 10 nucleotides in length.In other embodiments, the kits comprise antibodies to proteins encodedby the Steap3 nucleic acids. The kits of the present teachings can alsocomprise aptamers. The kit can contain in separate containers a nucleicacid or antibody (the antibody either bound to a solid matrix, orpackaged separately with reagents for binding to a matrix), controlformulations (positive and/or negative), and/or a detectable label, suchas but not limited to fluorescein, green fluorescent protein, rhodamine,cyanine dyes, Alexa dyes, luciferase, and radiolabels, among others.Instructions for carrying out the assay, including, optionally,instructions for generating a DAI score, can be included in the kit;e.g., written, tape, VCR, or CD-ROM. The assay can for example be in theform of a Northern hybridization or a sandwich ELISA as known in theart.

In some embodiments of the present teachings, Steap3 detection reagentscan be immobilized on a solid matrix, such as a porous strip, to form atleast one Steap3 detection site. In some embodiments, the measurement ordetection region of the porous strip can include a plurality of sitescontaining a nucleic acid. In some embodiments, the test strip can alsocontain sites for negative and/or positive controls. Alternatively,control sites can be located on a separate strip from the test strip.Optionally, the different detection sites can contain different amountsof immobilized nucleic acids, e.g., a higher amount in the firstdetection site and lesser amounts in subsequent sites. Upon the additionof test sample, the number of sites displaying a detectable signalprovides a quantitative indication of the amount of Steap3 in thesample. The detection sites can be configured in any suitably detectableshape and can be, e.g., in the shape of a bar or dot spanning the widthof a test strip.

In other embodiments of the present teachings, the kit can contain anucleic acid substrate array comprising one or more nucleic acidsequences. In some embodiments the substrate array can be on a solidsubstrate, such as what is known as a “chip.” See, e.g., U.S. Pat. No.5,744,305. In some embodiments the substrate array can be a solutionarray; e.g., xMAP (Luminex, Austin, Tex.), Cyvera (Illumina, San Diego,Calif.), RayBio Antibody Arrays (RayBiotech, Inc., Norcross, Ga.),CellCard (Vitra Bioscience, Mountain View, Calif.) and Quantum Dots'Mosaic (Invitrogen, Carlsbad, Calif.).

In some embodiments, the present invention is practiced using computerimplementation. In one embodiment, a computer comprises at least oneprocessor coupled to a chipset. Also coupled to the chipset are amemory, a storage device, a keyboard, a graphics adapter, a pointingdevice, and a network adapter. A display is coupled to the graphicsadapter. In one embodiment, the functionality of the chipset is providedby a memory controller hub and an I/O controller hub. In anotherembodiment, the memory is coupled directly to the processor instead ofthe chipset.

The storage device is any device capable of holding data, like a harddrive, compact disk read-only memory (CD-ROM), DVD, or a solid-statememory device. The memory holds instructions and data used by theprocessor. The pointing device may be a mouse, track ball, or other typeof pointing device, and is used in combination with the keyboard toinput data into the computer system. The graphics adapter displaysimages and other information on the display. The network adapter couplesthe computer system to a local or wide area network.

As is known in the art, a computer can have different and/or othercomponents than those described previously. In addition, the computercan lack certain components. Moreover, the storage device can be localand/or remote from the computer (such as embodied within a storage areanetwork (SAN)).

As is known in the art, the computer is adapted to execute computerprogram modules for providing functionality described herein. As usedherein, the term “module” refers to computer program logic utilized toprovide the specified functionality. Thus, a module can be implementedin hardware, firmware, and/or software. In one embodiment, programmodules are stored on the storage device, loaded into the memory, andexecuted by the processor.

Embodiments of the entities described herein can include other and/ordifferent modules than the ones described here. In addition, thefunctionality attributed to the modules can be performed by other ordifferent modules in other embodiments. Moreover, this descriptionoccasionally omits the term “module” for purposes of clarity andconvenience.

The following examples of specific aspects for carrying out the presentinvention are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way.

EXAMPLES Example 1: Identification of Steap3 Background

There is substantial donor-to-donor variability in the quality of humanRBC storage. Like humans, RBCs of different strains of inbred mice storedifferently; RBCs from C57BL/6 (B6) mice store well whereas RBCs fromFVB mice store poorly (24-hr post-transfusion recoveries).

Methods and Materials

B6×FVB F1 mice were interbred to generate an F2 population that has arandom assortment of chromosomes, including recombination events. F2mice were phenotyped by storing RBCs for 7 days, followed by transfusionand calculation of 24 hour recoveries. DNA from each F2 mouse wasanalyzed using a panel of 1400 single nucleotide polymorphisms (SNPs).Genetic elements associated with RBC post-transfusion survival wereidentified by Quantitative Trait Loci (QTL) analysis. Additionalresolution was obtained by backcrossing F2 mice with poor storage to B6parents, and selecting poor-storing progeny to breed for each nextgeneration.

Results

QTL analysis of 154 F2 mice revealed a peak signal on chromosome 1 atlocation rs4137908, with extreme statistical significance (p=2.09E-31).However, the peak was broad, and ranged over 149 Mb (using afalse-discovery rate of 0.05). SNP analysis of the backcrossed pedigreewas used to further refine the boundaries to a 9.5 Mb region, containing64 genes, 35 of which encoded proteins. Focusing on Non-Synonymous SNPsbetween B6 and FVB strains identified 5 genes (Gli2, Steap3, Ccdc93,Rab3gap1, and Tli). Based upon known biology, Steap3 was chosen as aleading candidate. Steap3 is the primary ferrireductase in erythroidcells, converting Fe3+ to Fe2+, both mitigating oxidative stress andallowing transferrin-dependent iron uptake. Mice lacking Steap3 haveprofound anemia. Moreover, a human family has been reported with anonsense mutation in the human steap3 orthologue and a congenitalhypochromic anemia. The SNP between B6 and FVB mice leads to an A-Vmutation at position 350, which is within the conserved ferric reductasesuperfamily domain.

Example 2: Steap3 Activity and RBC Storage

To test the ability of measuring Steap3 activity as a predictor of RBCstorage from genetically distinct donors, in a pre-clinical tractableanimal model, a variety of different strains of mice was characterizedfor RBC storage properties. Each strain had slightly different RBCstorage characteristics, as measured by post-transfusion 24-hourrecoveries of stored RBCs (FIG. 1A). 6 particular stains, ranging instorage, were chosen for further analysis by ferrozine assay (measuringSteap3 activity) (see FIG. 1B). A Pearson's correlation coefficient of−0.7185 was observed between the ferrozine activity and the 24-hr RBCrecoveries (FIG. 10). These data demonstrate, in a whole animal model,that measuring Steap3 activity predicts (as an inverse relationship) thepost-transfusion circulation of RBCs (e.g. 24-hour recovery) for donorsof different genetic composition.

REFERENCES

-   1. van de Watering L. Red cell storage and prognosis. Vox Sang 2011;    100: 36-45.-   2. van de Watering L. Pitfalls in the current published    observational literature on the effects of red blood cell storage.    Transfusion 2011; 51: 1847-1854.-   3. Fergusson D A, Hebert P, Hogan D L, LeBel L, Rouvinez-Bouali N,    Smyth J A, Sankaran K, Tinmouth A, Blajchman M A, Kovacs L, Lachance    C, Lee S, Walker C R, Hutton B, Ducharme R, Balchin K, Ramsay T,    Ford J C, Kakadekar A, Ramesh K, Shapiro S. Effect of fresh red    blood cell transfusions on clinical outcomes in premature, very    low-birth-weight infants: the ARIPI randomized trial. JAMA 2012;    308: 1443-1451.-   4. Lacroix J, Hebert P, Fergusson D, Tinmouth A, Blajchman M A,    Callum J, Cook D, Marshall J C, McIntyre L, Turgeon A F. The Age of    Blood Evaluation (ABLE) randomized controlled trial: study design.    Transfus Med Rev 2011; 25: 197-205.-   5. Steiner M E, Assmann S F, Levy J H, Marshall J, Pulkrabek S,    Sloan S R, Triulzi D, Stowell C P. Addressing the question of the    effect of RBC storage on clinical outcomes: the Red Cell Storage    Duration Study (RECESS) (Section 7). Transfus Apher Sci 2010; 43:    107-116.-   6. Hess J R. Red cell changes during storage. Transfus Apher Sci    2010; 43: 51-59.-   7. Hess J R. Red cell storage. J Proteomics 2010; 73: 368-373.-   8. Hod E A, Brittenham G M, Billote G B, Francis R O, Ginzburg Y Z,    Hendrickson J E, Jhang J, Schwartz J, Sharma S, Sheth S, Sireci A N,    Stephens H L, Stotler B A, Wojczyk B S, Zimring J C, Spitalnik S L.    Transfusion of human volunteers with older, stored red blood cells    produces extravascular hemolysis and circulating    non-transferrin-bound iron. Blood 2011; 118: 6675-6682.-   9. Hod E A, Spitalnik S L. Harmful effects of transfusion of older    stored red blood cells: iron and inflammation. Transfusion 2011; 51:    881-885.-   10. Hod E A, Spitalnik S L. Stored red blood cell transfusions:    Iron, inflammation, immunity, and infection. Transfus Clin Biol    2012; 19: 84-89.-   11. Hod E A, Zhang N, Sokol S A, Wojczyk B S, Francis R O, Ansaldi    D, Francis K P, Della-Latta P, Whittier S, Sheth S, Hendrickson J E,    Zimring J C, Brittenham G M, Spitalnik S L. Transfusion of red blood    cells after prolonged storage produces harmful effects that are    mediated by iron and inflammation. Blood 2010; 115: 4284-4292.-   12. Kor D J, Van Buskirk C M, Gajic O. Red blood cell storage    lesion. Bosn J Basic Med Sci 2009; 9 Suppl 1:21-27.-   13. Silliman C C, Moore E E, Kelher M R, Khan S Y, Gellar L, Elzi    D J. Identification of lipids that accumulate during the routine    storage of prestorage leukoreduced red blood cells and cause acute    lung injury. Transfusion 2011; 51: 2549-2554.-   14. Tissot J D, Rubin O, Canellini G. Analysis and clinical    relevance of microparticles from red blood cells. Curr Opin Hematol    2010; 17: 571-577.-   15. Dumont L J, AuBuchon J P. Evaluation of proposed FDA criteria    for the evaluation of radiolabeled red cell recovery trials.    Transfusion 2008; 48: 1053-1060.-   16. Gilson C R, Kraus T S, Hod E A, Hendrickson J E, Spitalnik S L,    Hillyer C D, Shaz B H, Zimring J C. A novel mouse model of red blood    cell storage and posttransfusion in vivo survival. Transfusion 2009;    49: 1546-1553.    While specific aspects of the invention have been described and    illustrated, such aspects should be considered illustrative of the    invention only and not as limiting the invention as construed in    accordance with the accompanying claims.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

The invention claimed is:
 1. A method for determining red blood cell(RBC) storage quality of an RBC unit, the method comprising: obtaining atest RBC sample from the RBC unit comprising Steap3 protein; contactingthe test RBC sample with a reagent that binds Steap3 protein to generatea complex of the reagent and Steap3 protein; detecting the complex toobtain RBC storage quality data associated with the RBC unit, whereinthe RBC storage quality data comprises a measured level of Steap3protein or activity in the test RBC sample, as compared to thecorresponding level in a control RBC sample suitable for transfusion;and storing the RBC storage quality data in a storage memory, whereinthe RBC storage quality data indicates that the RBC unit is suitable fortransfusion when the measured level of Steap3 protein or activity in thetest RBC sample is lower than or equal to the corresponding level in thecontrol RBC sample suitable for transfusion or wherein the RBC storagequality data indicates that the RBC unit is not suitable for transfusionwhen the measured level of Steap3 protein or activity in the test RBCsample is higher than the corresponding level in the control RBC samplesuitable for transfusion.
 2. The method of claim 1, wherein the RBCstorage quality data is obtained before storage of the RBC unit.
 3. Themethod of claim 1, wherein the RBC storage quality data is obtainedduring the time of storage of the RBC unit.
 4. The method of claim 1,wherein the RBC storage quality data is obtained by determining Steap3ferric reductase activity.
 5. The method of claim 1, further comprisingadministering the RBC unit to a subject if the RBC storage quality dataindicates that the RBC unit is suitable for transfusion.
 6. The methodof claim 1, further comprising not administering the RBC unit to asubject if the RBC storage quality data indicates that the RBC unit isnot suitable for transfusion.
 7. The method of claim 1, wherein thereagent is ferrozine and the detecting the complex comprises monitoringabsorbance of ferrous iron-ferrozine complex at λ=562 nm.
 8. The methodof claim 7, wherein the ferrozine is at 200 μM.
 9. The method of claim1, wherein the RBC storage quality data for the Steap3 marker comprisesSteap3 protein activity level in the test RBC sample, as compared to thecorresponding level in the control RBC sample suitable for transfusion,and the method further comprises determining post-transfusion survivalof RBCs by generating data on a level of at least one additionalbiomarker or indicator of blood storage in the test RBC sample, ascompared to the corresponding level in the control RBC sample suitablefor transfusion; and generating a score by mathematically combining theSteap3 protein activity level in the test RBC sample and the level ofthe at least one additional biomarker or indicator of blood storage inthe test RBC sample, wherein the score is indicative of post-transfusionsurvival of RBCs in the RBC unit, as compared to post-transfusionsurvival of RBCs in the control RBC sample suitable for transfusion. 10.The method of claim 9, wherein the score is used to determine whetherthe RBC unit will be administered to a subject.